Our long term objective is to understand the structure and function of eucaryotic chromosomes. This proposal delineates our future effort to study the functional role of a particular DNA topoisomerase, the ATP-dependent topoisomerase from Drosophila melanogaster, in this general area. The Drosophila ATP-dependent topoisomerase is able to catalyze the following three topoisomerization reactions: relaxation of superhelical DNA, catenation and decatenation of duplex DNA rings. The mechanism of this enzyme involves the passage of a DNA segment through a transient double-strand break, during which process the broken ends are not allowed to rotate with respect to each other. We will first study the mechanism of this novel enzyme in order to have a mechanistical understanding of how the enzyme can pass one segment of DNA through another one. We will isolate the intermediate of the enzyme covalently linked to the ends of the enzyme-cleaved site and investigate the structure of the cleavage site. The search for a novel intermediate in which one DNA helix is topologically linked to another one will shed light on the question whether the enzyme works in a sequential manner to transport DNA helices through each other. We will also attempt to study the function of this enzyme with our attention primarily focused on initiation of DNA replication, condensation and decondensation of eucaryotic chromosomes. Establishing a soluble, in vitro DNA replication system from Drosophila cleavage embryos which are known to undergo very rapid DNA synthesis will enable us to ask whether this enzyme is involved in the initiation of DNA replication. We will use the catenation to study how this enzyme might condense the chromosome. Various basic proteins, including histones H1 and its phosphorylated form, will be isolated and their effect on the catenation of DNA and chromatin by this enzyme will be investigated.